Inhibitory Serpins
Serpins are irreversible serine protease inhibitors which are principally located extracellularly. As a group, they are defined on the basis of their structural and functional characteristics: a high molecular weight (between 370-420 amino acid residues), and a C-terminal reactive region. Proteins which have been assigned to the serpin family include the following: .alpha.-1 protease inhibitor, .alpha.-1-antichymotrypsin, antithrombin III, .alpha.-2-antiplasmin, heparin cofactor II, complement C1 inhibitor, plasminogen activator inhibitors 1 and 2, glia derived nexin, protein C inhibitor, rat hepatocyte inhibitors, crmA (a viral serpin which inhibits interleukin 1-.beta. cleavage enzyme), human squamous cell carcinoma antigen which may modulate the host immune response against tumor cells, human maspin which seems to function as a tumor suppressor, lepidopteran protease inhibitor, leukocyte elastase inhibitor (the only known intracellular serpin), and products from three orthopoxviruses (these products may be involved in the regulation of the blood clotting cascade and/or of the complement cascade in the mammalian host).
Serpins form tight complexes with their target proteases. The serpin region which binds to the target protease is a mobile, exposed reactive site loop (RSL) which contains the P1--P1' bond that is cleaved. When the characteristic serpin P1--P1' bond cleaves, the serpin structure changes profoundly, and stability to heat- or guanidine-induced denaturation increases markedly. These changes are referred to as the stressed-to-relaxed (S.fwdarw.R) transition, and are associated with tight complex formation with specific proteases. For the .alpha.1-proteinase inhibitor, cleavage of the P1--P1' bond results in a separation of about 69 .ANG. between the two residues (Loebermann H et al (1984) J Mol Biol 177:531-556). The ability of a serpin to function as an inhibitor may be directly related to its ability to undergo this S.fwdarw.R transition (Bruch M et al (1988) J Biol Chem 263: 16626-30; Carrell RW et al (1992) Curr Opin Struct Biol 2:438-446).
In addition, the RSL sequence from P17 to P8 (hinge region) is highly conserved, and small amino acid with side chains are found at positions P9, P10, P11, P12, and P15 in active inhibitors. The presence of small amino acids in this region allows the peptide loop from P14-P2to be inserted into the middle of the protease inhibitor A-sheet. The insertion of this sequence into the A-sheet appears to be important in stabilizing the inhibitor, and consequently tightening the protease/serpin complex. Sequence divergence in the hinge region may convert an inhibitor to a substrate.
Noninhibitory Serpins
A number of proteins with no known inhibitory activity are also categorized as serpins on the basis of strong sequence and structural similarities. These proteins can be cleaved by specific proteases, but do not form the tight complexes that inhibit protease activity. Examples are bird ovalbumin, angiotensinogen, barley protein Z, corticosteroid binding globulin, thyroxine binding globulin, sheep uterine milk protein, pig uteroferrin-associated protein, an endoplasmic reticulum heat-shock protein (which binds strongly to collagen and could act as a chaperone), pigment epithelium-derived factor, and an estrogen-regulated protein from Xenopus.
The nature of the difference between inhibitory and noninhibitory serpins is not well understood. For example, ovalbumin is unable to undergo this S.fwdarw.R transition (Mottonen et al (1992) Nature 355: 270-273). However, hormone binding globulins, such as thyroxine or cortisol binding globulins, apparently do undergo the transition from the native stressed to relaxed conformation upon protease cleavage but do not form a tight complex with specific proteases (Pemberton et al (1988) Nature 336: 257-258). The S.fwdarw.R transition may confer an advantage for hormone binding molecules, and for small molecule binding proteins in general, in that the transition from a stressed to a relaxed conformation may provide a method for modulating hormone delivery. Both hormone binding globulins have a greater than 30% homology with the archetype of the serpin family, alpha-1-antitrypsin, and sequence matching infers that they all share a common secondary and tertiary structure.
Serpins are defined and described in Carrell R and Travis J (1985) Trends Biochem Sci 10:20-24; Carrell R et al (1987) Cold Spring Harbor Symp Quant Biol 52:527-535; Huber R and Carrell R W (1989) Biochemistry 28:8951-8966; and Remold-O'Donneel E (1993) FEBS Lett 315:105-108.
The novel serpin which is the subject of this application was identified among the cDNAs of a pooled hypothalamus library.
The Hypothalamus
The hypothalamus, the master gland of the human body, is an area of neuroendocrine cells on the floor and midline of the human brain. It is intimately associated with the nervous system function. The anterior hypothalamus mostly interacts with parasympathetic pathways, and the posterior with sympathetic. Functionally, the hypothalamus is divided into chiasmatic, tuberal and mammillary regions.
The chiasmatic region which develops prenatally has three prominent components, the supraoptic, paraventricular and accessory neurosecretory nuclei; the sexually dimorphic intermediate nucleus (SDN); and the suprachiasmatic nucleus (SCN). The large supraoptic (SON) and paraventricular neurons (PVN) of the chiasmatic region are unmyelinated and produce antidiuretic hormone (ADH) and oxytocin; the enzyme, tyrosine hydroxylase; and the monoamine neurotransmitter, dopamine. Subsequently, ADH and oxytocin are stored in the anterior pituitary gland. PVN neurons also produce somatostatin. The neurosecretory granules of these large-celled neurons produce small amounts of dynorphin, enkephalins, galanin, cholecystokinin, and neuropeptide Y which appear to function as local paracrine agents.
The small accessory neurosecretory neurons produce ADH, tyrosine hydroxylase, neuropeptide Y, and corticotropin releasing hormone (CRH) and have prominent dopamine synapses. Neurons of the chiasmatic region contain gamma amino butyric acid (GABA), glutamate, quisqualate, relaxin, melatonin, angiotensin-1, endothelin, N-methyl-D-aspartate (NMDA), neurophysin, and .beta.-adrenergic receptors (Morris JF and Pow DV (1993) Ann NY Acad Sci 689:16-33; Renaud LP et al (1992) Prog Brain Res 92:277-288). Projections of all three types of chiasmatic neurons communicate with many regions of the central nervous system including the brain stem, limbic system, retina and spinal cord.
The sexually dimorphic nucleus (SDN), also known as the intermediate nucleus, is located between the supraoptic and paraventricular nuclei. The SDN appears to be sensitive to steroidal hormones and develops twice as many cells and is twice as large in males (0.2 mm.sup.3) as in females (0.1 mm.sup.3) after the age of four. The number of cells decreases with senescence (at 50 years of age for men and 70, for women); however, cause and effect of associated hormones have not been established.
The superchiasmatic nucleus (SCN) is considered to be the circadian pacemaker of the mammalian brain coordinating both hormonal and behavioral rhythms. The SCN is sexually dimorphic, elongated in women and spherical in men; and the number and volume of SCN cells varies with age and season. Biochemical and immunological studies indicate that serotonin and melanin in concert with G-protein associated/cyclic adenosine monophosphate-linked receptors regulate circadian rhythms (Erlander MG et al (1993) J Biol Rhythms 8S:25-31).
The retinohypothalamic tract is a monosynaptic pathway that links the retina to the SCN and helps set the intrinsic period, phase, and amplitude of the internal biological clock. Total blindness which prevents light/dark synchronization results in free-running rhythms, particularly in cortisol, melatonin, sleep, and temperature regulation. A lesion or tumor in the area of the SCN can also be correlated with disturbed circadian rhythms.
The hypothalamic neurons of the tuberal and mammillary regions produce a variety of regulatory peptides called releasing hormones or factors which modulate much of human endocrine function. These short oligopeptide releasing factors are secreted and delivered to the anterior pituitary via the fenestrated capillary network of the hypothalamic pituitary portal system. Each region will be discussed in turn.
The tuberal region is composed of a complex of ventromedial (VMN), dorsomedial (DMN), lateral tuberal (NTL), and infundibular nuclei (Braak, H and Braak, E (1992) Prog Brain Res 93:3-14). These nuclei function in feeding, aggressive and sexual behaviors, and they secrete growth hormone releasing hormone (GRH), thyroid releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH). The networked VMN has projections to the basal forebrain as well as to all parts of the cerebral cortex where it is assumed to influence higher cortical function.
The DMN is poorly differentiated in the human brain and covers the anterior and superior areas of the VMN. Its neurons contain catecholamine, somatostatin, neuropeptide Y and neurotensin, neurokinin B (NKB), and LHRH. The NKB neurons may participate negative feedback of estrogen on LHRH and act as an intemeuron on LHRH nuclei.
The NTL is only present in higher primates. The NTL is characterized by cholinergic, CRH, somatostatin, benzodiazepin and NMDA receptors. Neuronal loss in this region may predict disease severity, particularly in Kallmann's and Down's syndromes and in Alzheimer's and Huntington's diseases.
The exact role of the mammillary nucleus is poorly defined. Most of its neurons project into the cortex and are responsible for the major histaminergic innervation. Some evidence indicates the mammillary region is involved in heat regulation and governs capillary restriction, sweating, shivering and piloerection.
Nonendocrine functions of the hypothalamus include regulation of food intake and feeding behavior, temperature regulation, sleep-wake cycle, memory, behavior, and thirst. Although the basal hypothalamus is known to control stable weight, both the VMN and the anterior hypothalamus are involved in regulation of hunger and satiety. Appetite is stimulated by GABA, dopamine, beta-endorphins, enkephalin and neuropeptide Y and inhibited by serotonin, norepinephrine, cholecystokinin, neurotensin, TRH, naloxone, somatostatin, and vasoactive intestinal peptide. A lesion or tumor in the area of the VMN can cause hypothalamic obesity. Other factors, particularly the thyroid and adrenal hormones, also affect eating behavior.
The anterior hypothalamus contains neurons that respond to local and environmental thermal gradients. Heat production is stimulated by serotonin and blocked by norepinephrine and epinephrine. When infections occur, phagocytic cells produce interleukin-1 (IL-1). IL-1 stimulates the anterior hypothalamus to produce prostaglandin E2 which increases the body temperature set point and produces fever. Cooling or heat dissipation which involves vasodilation is governed by the posterior hypothalamus. Hypothalamic disease (with or without malfunction of the thyroid or adrenal glands) may cause hypothermia, hyperthermia or poikilothermia.
The sleep center is located in the anterior hypothalamus where disturbances or lesions can lead to insomnia or agitation. The posterior hypothalamus is responsible for arousal and maintenance of the waking state. Serotonin promotes sleep, while catecholamines aid wakefulness. Destruction of the posterior hypothalamus, for example, by ischemia and encephalitis, trauma or tumor can result in hypersomnolence.
Thirst is controlled by serum osmolality and is detected by osmoregulators in the hypothalamus. Nerve impulses control the pituitary release of vasopressin which acts upon the kidney. In the case of pathological disturbances, interactions among the nervous system, endocrine hormones, and cytokines (particularly IL-1) modulate the activity of these glands and the kidney. Impaired thirst is commonly attributable to hypothalamic lesions.
The effects of ACTH, ADH, and oxytocin memory and behavior are still being investigated. Lesions of the ventromedial hypothalamus produce rage while ventromedial, dorsomedial and/or mammillary lesions cause loss of short term memory. Lateral hypothalamic destruction can cause apathetic behavior, but large hypothalamic lesions are associated with dementia.
Diseases Associated with the Hypothalamus
Many diseases are associated with changes in hypothalamic function and structure.
The most common hypothalamic disease is hyperprolactinemia, excess prolactin production, which may lead to galactorrhea and/or hypogonadism. Another disease is dwarfism, likely caused by the overproduction of somatostatin which prevents growth hormone release. Tumors are the most common cause of the over- or under-production of hypothalamic hormones. Cushing's disease is caused by tumors overproducing ACTH. Finally, the hypothalamus or the molecules it produces may also be responsible for some of the symptoms in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases.
Hypothalamic anatomy, physiology, and diseases are reviewed, inter alia, in Guyton AC (1991) Textbook of Medical Physiology, WB Saunders Co, Philadelphia Pa.; Isselbacher KJ et al (1994) Harrison's Principles of Internal Medicine, McGraw Hill, New York City; The Merck Manual of Diagnosis and Therapy (1992) Merck Research Laboratories, Rahway N.J.; and Swaab DB et al (1993) Anat Embryol 187:317-330.
Some of these diseases may be difficult to diagnose or treat. Modem techniques for diagnosis of abnormalities in the hypothalamus mainly rely on observation of clinical symptoms, serological analysis of hormone levels, or measurement of urinary excretion of a hormone or its metabolites. Alternatively, computerized axial tomography (CAT scan) or Magnetic Resonance Imaging (MRI) can be used to observe abnormal histological changes of the hypothalamic region. Thus, development of new techniques becomes necessary for early and accurate diagnosis or for treatments of diseases associated with the hypothalamus.